Virus assembly is the final step in the life cycle of a virus prior to its release from the infected cell. Detailed information concerning this process is fundamental for the development of more effective antiviral strategies and will be useful in developing viral vectors that can be used in genetic engineering and medicine. The proposed project will investigate the assembly of alphaviruses. The aiphaviruses are a group of arthropod-borne, plus-strand RNA viruses, many of which cause encephalitis, arthritis, myositis and fever in humans. They represent one of the simplest types of enveloped animal viruses and are model systems for assembly, in part, due to the well-defined structural organization of the virion. We propose three aims: 1) to investigate the process by which these viruses interact with their genome RNA to specifically package it inside the nucleocapsid core, 2) to describe the process by which the core assembles into spherical particles, and 3) to examine how the outer glycoproteins organize into an icosahedral lattice and impose this symmetry on the inner nucleocapsid core. A multi-disciplinary approach will use molecular genetics, biochemistry and structural techniques to probe the mechanism of virus assembly and to ultimately describe the process in atomic detail. Using cryo-electron microscopy image reconstructions of the whole virus, and the atomic structure of the nucleocapsid protein and possibly the El and E2 glycoproteins as a guide, site-directed mutagenesis will be carried out to probe structure-function relationships. The resulting mutants will be studied by a variety of in vivo and in vitro biochemical assays that will examine protein-protein and protein-nucleic acid interactions involved in nucleocapsid core formation and interactions of the nucleocapsid core with the icosahedral-organized glycoproteins. Biophysical techniques such as x-ray crystallography, nuclear magnetic resonance and cryo-electron microscopy together with image reconstruction will examine wild type and mutant proteins involved in the assembly process. The proposed research will advance our knowledge of virus assembly, macromolecular interactions, and serve as a paradigm for the molecular mechanism of virus and cellular budding.